4,4'-methylenebis(cyclohexylamine)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

GLP guideline study (OECD 443, rat) of the structural analogue DMDC (CAS 6864-37-5). PACM and DMDC are considered read-across analogues based on structural similarity and similar physico-chemical and toxicological properties. The common structural features of the two substances are: a common functional primary amine group; the amines are bound to a cyclic aliphatic organic substituent; there are no elements other than carbon, hydrogen and nitrogen; identical structures except for a methyl group on each cyclohexane, ortho to the amine on DMDC similar molecular weights, both under 500 daltons, qualifying as “low molecular weight” compounds. A read-across approach according to Regulation (EC) No 1907/2006 (REACH) Annex XI 1.5 and following ECHA Read-Across Assessment Framework, RAAF (2015) is attached in the respective IUCLID chapters.

PACM is registered under REACH Annex X with identified data gaps at the endpoint toxicity to reproduction. A study to check for fertility influencing effects as well as a study for pre-natal developmental toxicity (second species) are missing. However, these data are available for the analogue chemical DMDC. For this purpose, those data will be used as a source to fill existing data gaps (e.g. OECD 443 study).

The data of the EOGRT study (OECD 443) with DMDC have been used for derivation of systemic DNELs.

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

GLP guideline study (OECD 443, rat) of the structural analogue DM-PACM (CAS 6864-37-5). PACM and DM-PACM are considered read-across analogues based on structural similarity and similar physico-chemical and toxicological properties. The common structural features of the two substances are: a common functional primary amine group; the amines are bound to a cyclic aliphatic organic substituent; there are no elements other than carbon, hydrogen and nitrogen; identical structures except for a methyl group on each cyclohexane, ortho to the amine on DM-PACM similar molecular weights, both under 500 daltons, qualifying as “low molecular weight” compounds. For a detailed read across justification see also the attached justification document.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

1.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

haematology

clinical biochemistry

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

1.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/kg bw/day (nominal)

Organ:

heart

kidney

lungs

lymph node

pancreas

pituitary gland

seminal vesicle

stomach

other: esophagus, skeletal muscle, left epididymidis

Treatment related:

yes

Dose response relationship:

yes

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1A)

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1A)

Effect level:

1.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1B)

Effect level:

1.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 2A)

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

neuropathology

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 2B)

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

neuropathology

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 3)

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental immunotoxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/kg bw/day (nominal)

Organ:

heart

kidney

lungs

lymph node

pancreas

seminal vesicle

other: eyes, esophagus

Treatment related:

yes

Dose response relationship:

yes

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

5 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1.5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

modern OECD 443 study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

GLP guideline study (OECD 443, rat) of the structural analogue DMDC (CAS 6864-37-5) [BASF, 2020].

PACM and DMDC are considered read-across analogues based on structural similarity and similar physico-chemical and toxicological properties. The common structural features of the two substances are: a common functional primary amine group; the amines are bound to a cyclic aliphatic organic substituent; there are no elements other than carbon, hydrogen and nitrogen; identical structures except for a methyl group on each cyclohexane, ortho to the amine on DM-PACM similar molecular weights, both under 500 daltons, qualifying as “low molecular weight” compounds. A read-across approach according to Regulation (EC) No 1907/2006 (REACH) Annex XI 1.5 and following ECHA Read-Across Assessment Framework, RAAF (2015) is attached in the respective IUCLID chapters.

 

PACM is registered under REACH Annex X with identified data gaps at the endpoint toxicity to reproduction. A study to check for fertility influencing effects (OECD 443) as well as a study for pre-natal developmental toxicity (second species) are missing (OECD 414). However, these data are available for the analogue chemical DMDC. For this purpose, those data will be used as a source to fill existing data gaps (e.g. OECD 443 study).

 

The data of the EOGRT study (OECD 443) with DMDC have been used for derivation of systemic DNELs:

The NOAEL (no observed adverse effect level) for general, systemic toxicity is the low dose of 1.5 mg/kg bw/d for the F0 and F1 animals. This was based on treatment-related, adverse effects such as a reduction in water and food consumption, decrease in body weight (change), altered clinical pathology parameters as well as histopathological changes in several organs, which were observed at the high- and mid-dose of 15 and 5 mg/kg bw/d. The NOAEL for fertility and reproductive performance for the F0 parental rats is 1.5 mg/kg bw/d, the lowest dose tested. This was based on the lower mean number of implantation sites and secondary decreased mean number of F1 pups delivered per dam in the high- and mid-dose groups. The NOAEL for developmental toxicity in the offspring is the mid-dose of 5 mg/kg bw/d, based on the decrease in pup body weight during lactation at the high-dose level of 15 mg/kg bw/d. This slight delay in development of the high-dose pups was observed in presence of maternal toxicity and, therefore, not assessed as independent effect. The NOAEL for developmental neurotoxicity in the F1 progeny is 15 mg/kg bw/d, the highest dose tested. Neuropathological findings observed in Cohort 2A adults of high- and mid-dose indicated a systemic, direct toxicity of the test substance and were not assessed as developmental neurotoxicological effects. The NOAEL for developmental immunotoxicity for the F1 progeny is 15 mg/kg bw/d, the highest dose tested. Lower mean and median anti-SRBC IgM antibody titers of the positive control group (4.5 mg/kg bw/d cyclophosphamide, oral) demonstrated that the test system worked properly.

 

Both substances were tested in OECD 422 repeated dose/reproductive toxicity screening tests on rats using very similar dose settings with the result that entirely the same NOAEL was deduced. Those two studies were used as bridging studies to proof the same toxicological profile of both substances. At 15 mg/kg bw none of the available data give any hint on a potential of PACM as well as DMDC to influence the reproductive performance and fertility or the offspring’s development. Therefore, it is assumed that the health effects, especially potential fertility influencing effects, may be predicted from the available data for the source substance at this specific endpoint (OECD 443).

 

Briefly, in a study according to OECD 422, marked as supporting information, DMDC has been administered by oral gavage to Wistar rats. Signs of systemic toxicity at the highest dose of 15 mg/kg bw/d have been observed with regard to a reduction in food consumption and decrease in body weight (change), adverse changes in clinical pathology parameters and histopathology. No test-item related effects on oestrus cycle, sperm measures and reproductive performance were observed and therefore, the NOAEL for reproductive performance and fertility was set to 15 mg/kg bw/d for male and female Wistar rats. Furthermore, no treatment-related effects on the foetuses up to PND 13 were detected and the NOAEL for developmental toxicity was 15 mg/kg bw/d, the highest dose tested. [BASF, 2019]

 

4,4’-Methylenedicyclohexanamine (PACM) was examined in rats in an OECD 422 (28 -day repeated dose toxicity/reproductive toxicity) screening guideline study at doses of 15, 50 and 100 mg/kg bw/d. This study is also classified as supporting information. No abnormalities were seen in the reproductive organs of either fertile or non-fertile animals. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impairment. Slight reproductive toxicity was noted as a decrease in implantation sites at the 50 and 100 mg/kg bw/d dose levels, the same dose at which systemic toxicity was noted in repeated dose oral toxicity studies. The control group had a number of implantation sites slightly higher than historical controls. At the lower of these two doses, there were two of 9 females with no implantation sites, which may have occurred by chance. This led to a reduction in the gestation index at 50 mg/kg bw/d, but not at 100 mg/kg bw/d. The lack of dose response causes the relevance of this parameter to be questions. [NOTOX, 2010]

 

Final discussion

In the OECD 443 with DMDC treatment-related, adverse effects such as a reduction in water and food consumption, decrease in body weight (change), altered clinical pathology parameters as well as histopathological changes in several organs were observed at the high- and mid-dose of 15 and 5 mg/kg bw/d. Concerning developmental neurotoxicity and immunotoxicity, there were no findings up to the highest dose tested. In the mid dose group, a slight delay in development (decreased pub body weight during lactation) was seen, assessed as a non-independent, secondary effect as a consequence of maternal toxicity. Concerning fertility and reproductive performance for the F0 parental rats, the NOAEL was set to 1.5 mg/kg bw/d, the lowest dose tested. This was based on a lower mean number of implantation sites and secondary decreased mean number of F1 pups delivered per dam in the high- and mid-dose groups. All effects were only observed in connection with pronounced systemic effects and therefore considered not relevant in terms of classification and labelling.

Effects on developmental toxicity

Description of key information

To be compliant with the REACH Regulation, two pre-natal developmental toxicity studies need to be submitted for a substance registered under REACH Annex X.

For the registered substance (PACM) an OECD 414 with rats is available [Envigo, 2016], but an OECD 414 using a second species is not. However, a GLP guideline study (OECD 414, rabbit) of the structural analogue DMDC (CAS 6864-37-5) is on hand. [BASF, 2017]

PACM and DMDC are considered read-across analogues based on structural similarity and similar physico-chemical and toxicological properties. The common structural features of the two substances are: a common functional primary amine group; the amines are bound to a cyclic aliphatic organic substituent; there are no elements other than carbon, hydrogen and nitrogen; identical structures except for a methyl group on each cyclohexane, ortho to the amine on DMDC similar molecular weights, both under 500 daltons, qualifying as “low molecular weight” compounds. A read-across approach according to Regulation (EC) No 1907/2006 (REACH) Annex XI 1.5 and following ECHA Read-Across Assessment Framework, RAAF (2015) is attached in the respective IUCLID chapters.

PACM is registered under REACH Annex X with identified data gaps at the endpoint toxicity to reproduction. A study to check for fertility influencing effects as well as a study for pre-natal developmental toxicity (second species) are missing. However, these data are available for the analogue chemical DMDC. For this purpose, those data will be used as a source to fill existing data gaps (e.g. OECD 414 study).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

GLP guideline study (OECD 414, rabbit) of the structural analogue DM-PACM (CAS 6864-37-5). PACM and DM-PACM are considered read-across analogues based on structural similarity and similar physico-chemical and toxicological properties. The common structural features of the two substances are: a common functional primary amine group; the amines are bound to a cyclic aliphatic organic substituent; there are no elements other than carbon, hydrogen and nitrogen; identical structures except for a methyl group on each cyclohexane, ortho to the amine on DM-PACM similar molecular weights, both under 500 daltons, qualifying as “low molecular weight” compounds. For a detailed read across justification see also the attached justification document.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Effect level:

9 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: highest dose tested

Key result

Developmental effects observed:

no

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
-Species: Hannover Wistar Rat
-Supplier: Charles River Laboratories, Germany
-Breeder: Charles River Laboratories, Germany, Sandhofer Weg 7, 97633 Sulzfeld, Germany
-Number: 88 females (the females were nulliparous and non-pregnant), 30 males
-Total number of animals ordered: 100 females and 30 males (spare animals was used as needed and their exchange and/or sacrifice was documented in the raw data).
-Total number of females per group: 22
-Age (at delivery): Approximately 10-11 weeks
-Female body-weight range (day 0 post coitum): 191-239 g

CONDITIONS
-Standard laboratory conditions: air-conditioned with a minimum of 15-20 air changes per hour; the environment monitored continuously with target ranges for temperature of 22 ± 2 °C and for relative humidity between 40 and 70%, 12 hours artificial fluorescent light (7:00 am to 7:00 pm)/ 12 hours dark (7:00 pm to 7:00 am). Values outside these ranges occasionally occurred. These transient variations were considered not to have any influence on the study.

-Accommodation: Cages with Lignocel S8-15 sawdust bedding (J. Rettenmaier & Söhne).
-Females:
Acclimatization period (maximum 5 animals/cage): Makrolon cages -IV
Pregnancy (individually): Makrolon cages -III
-Males:
Acclimatization and pairing (individually): Makrolon cages –III
Acclimatization: 5 days

-Diet: Pelleted standard Teklad 2014C rat/mouse maintenance diet ad libitum (supplied by Envigo RMS, S.L., batches no.: 120415MA and 121415MA, expiry dates: 30 August and 09 September 2016, respectively).
The diet was analyzed by the manufacturer for contaminants and to check its composition. The results of analyses will be archived at Envigo CRS, S.A.U.
Water: Tap water in bottles ad libitum.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of dose formulation was determined twice during the study and once (top/middle/bottom) for homogeneity in samples taken from the formulation administered to Groups 2 to 4 by GC. The formulations were quantified following the analytical procedure validated in the present study and using the computerized system EZ Chrom.
The test item was used as analytical standard.
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The homogeneity and stability was confirmed for the test item in vehicle formulations at nominal concentrations of 1, 3 and 10 mg/mL when stored refrigerated for 5 days.
The mean concentrations of test item in test formulations analyzed for the study were within 100 ± 10 % of nominal concentrations, confirming accurate formulation.

Details on mating procedure:

After acclimatization, females were housed with sexually mature males in a cage for a period approximately of 16 hours (maximum 2:1). Vaginal smears were taken daily until mating is confirmed. The females were removed and housed individually when:
The vaginal smear was sperm positive, or A copulation plug was observed.
The day of mating was designated day 0 post coitum.
Male rats of the same source and strain were used for mating only. Clinical signs and body weight were recorded weekly but do not report. Data are included in the raw data.

Duration of treatment / exposure:

The females received the test item from days 6 to 19 post coitum.

Frequency of treatment:

Once daily, Administration volume 5 mL/kg

Duration of test:

20 days, The females received the test item from days 6 to 19 post coitum.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

low dose group

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

intermediate dose group

Dose / conc.:

50 mg/kg bw/day

Remarks:

high dose group

No. of animals per sex per dose:

Group 1 2 3 4
Dose Control 5 mg/kg/day 15 mg/kg/day 50 mg/kg/day
Total no. of females 22 22 22 22

Total number of males : 30

Control animals:

yes, concurrent vehicle

Details on study design:

-Species: Recognized by international guidelines as a recommended test system.
-Method: This route is an anticipated route of human exposure to the test item.
-Dose levels: The dose levels were selected by the Sponsor in view of the results
finding in previous studies.

Maternal examinations:

All females sacrificed during the study were subjected to macroscopic examination. Postmortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea was performed and the data recorded. The uteri (and contents) of all females was weighed during necropsy on day 20 post coitum to enable the calculation of the corrected body-weight gain.

Fetal examinations:

Fetuses and placenta were removed from the uterus and weighed individually. Fetuses were sexed, examined for gross external abnormalities (Makris, 2009), sacrificed by intraperitoneal injection of sodium pentobarbital and allocated to one of the following procedures:
1. One-half of the fetuses from each litter were fixed in Bouin's fixative (Wilson, 1965). They were serially sectioned and examined (evaluation of the internal structures of the heads, thoracic and abdominal organs). The tissue was preserved in a solution of 96% ethyl alcohol (one fetus per container). Descriptions of any abnormalities and variations were recorded.
2. The remaining fetuses were eviscerated and fixed in 70% alcohol. Then they were placed in a solution of potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and a solution of glycerin/alcohol for preservation and storage (Dawson, 1926). The skeletons were examined and all abnormal findings and variations were recorded. The specimens were individually preserved.
When considered appropriate, macroscopic changes in the fetuses were photographed and if necessary samples of tissue fixed in a suitable fixative for possible microscopic examination. The photographs will be archived with the raw data.
The fetuses were shipped (on 09 August 2016) to the PI (Debbie Coulby, Envigo CRS Limited) for evaluation. The necessary necropsy/cesarean data previously recorded in Pristima System was entered manually in the validated VMS System. No Phase number was assigned; CB24RV is used as study identifier.

Statistics:

The following comparisons were performed:
Group 1 vs. Groups 2, 3 and 4 to analyze food consumption, body weights, clinical signs and reproduction data:
Means and standard deviations of various data were calculated and included in the report.
For continuous data, a parametric analysis was performed if Bartlett's test for variance homogeneity is not significant at the 1% level. Treated groups were compared to control using Williams' test, unless there is evidence against a monotonic dose-response when Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test is still significant at the 1% level following both logarithmic and square-root transformations. Treated groups were compared to control using Shirley's test, unless there is evidence against a monotonic dose-response when Steel's test was performed instead.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were recorded.
Some findings as coat, hair loss (dorsal or ventral surface, forelimbs and hind limbs) were observed sporadically in all the treatment groups.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slightly lower mean body weight was recorded at the doses of 50 and 15 mg/kg/day compared to the control group from days 11 and 17 post coitum onwards, respectively. Regarding body-weight gain, statistically significant differences were recorded at the above mentioned doses, from days 11 to 20 of pregnancy at 50 mg/kg/day and from days 18 to 20 of pregnancy at 15 mg/kg/day.
No differences compared to the control group were recorded at the dose of 5 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A dose-dependent statistically significant decrease in absolute food consumption (gram per animal per day) compared to the control group was recorded at the doses of 50 and 15 mg/kg/day and on days 9 to 20 and 12 to 18 of pregnancy, respectively.
Food consumption at the dose of 5 mg/kg/day was not affected by the treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test-item-related findings were recorded in the macroscopic examination at hysterectomy in the rats treated with the test item.
Some findings were recorded among all groups, including control: dilated renal pelvis, pale and/or strangulated lobe of liver, thin wall of diaphragm, split cervical thymus.
These findings were not attributed to the test item and were considered to be within the range of normal background lesions that may be seen in rats of this strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

There were no relevant test-item related changes.
Differences were recorded in some reproductive parameters. The statistically significant differences observed were devoid of any toxicological relevance. They were considered a consequence of the biological variability and not test-item related.
There were no differences between the left and the right side concerning the distribution of resorptions in the uterine horns nor in the total resorptions nor in the uterus and placenta weight.

Key result

Dose descriptor:

NOAEL

Effect level:

> 50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Based on the results of this study, the dose of 50 mg/kg/day is considered the dose with NOAEL for pregnant females. The slightly lower bodyweight increase recorded at this dose could be considered as an indication of a minimal toxic effect.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slight higher fetal weights (males) were recorded at 15 and 50 mg/kg/day, approximately 9% and 6%, respectively, when compared with the control group. The differences observed were statistically significant.
No differences compared to the control group were recorded at 5 mg/kg /day or in the females at any of the treated doses

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No differences in the percentage of male/female pups ratio were noted.

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings which could be attributed to the treatment were observed.
50 mg/kg/day
Litter no. 104: One fetus (no.11) presented left hind paw with restricted movement
15 mg/kg/day
Litter no. 68: One fetus (no. 1) had domed head and short upper jaw
Litter no. 70: One fetus (no. 4) had cleft palate

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 50 mg/kg/day there was a slightly increased incidence of medially thickened/kinked ribs, misaligned sternebral ossification sites and ossified cervical vertebral centra compared to control group. These findings are considered as minor skeletal abnormalities.
No relevant findings were recorded at 15 or 5 mg/kg/day.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Some isolated major abnormalities were recorded among the doses 5 and 15 mg/kg/day.
One fetus (no. 4) from litter 50 treated at 5 mg/kg/day presented right sided esophagus; right sided aortic arch; narrow, dorsally displaced retroesophageal pulmonary trunk; diaphragmatic hernia; membranous septal defect ventricular septum; malrotated heart; large posterior caudate liver lobe.
One fetus (no. 1) from litter 75 treated at 15 mg/kg/day presented ascending aorta pulmonary trunk fistula, large right ventricle, large heart accompanied by left umbilical artery, posterior caudate liver lobe fissure.
No major findings were recorded at 50 mg/kg/day. Some minor visceral abnormalities were observed in all the groups, including control (partially undescended lobe of thymus, left umbilical artery).

Key result

Dose descriptor:

NOAEL

Effect level:

> 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

Remarks on result:

other: Despite the presence of abnormalities at external and visceral examination at the low and intermediate doses, there was no evidence that the test item could significantly increase the occurrence of teratogenic effects.

Key result

Developmental effects observed:

no

Conclusions:

The test item, 4,4'-methylenebis(cyclohexylamine) was administered once daily by oral gavage to three treatment groups of twenty two pregnant female Wistar rats each from day 6 to day 19 of gestation at dose levels of 5, 15 and 50 mg/kg body weight/day. A control group of twenty two females were administered with vehicle (propyleneglycol) alone. The treatment with test item resulted in no mortalities. All the dams survived till the scheduled sacrifice. No test item related clinical signs were observed in any of the pregnant female animals of low, intermediate and high dose groups as well as in control group. The body weight, body weight gain (%) and feed consumption during gestation period in all treatment groups were comparable with control group. No treatment-related effects were observed in reproduction parameters. The external and visceral variations observed in foetus were not statistically significant. Therefore these findings were considered as incidental findings and not test item-related. Based on the study findings, it is considered that the test item is non teratogenic in rats and the NOAEL for teratogenicity of the test item can be fixed as 50 mg/kg bw/day, under the present experimental conditions. The NOAEL for maternal toxicity was considered as 50 mg/kg bw/day.

Executive summary:

The test item PACM (Curing Agent) was administered by oral gavage, from day 6 post coitum to day 19 post coitum, inclusive, at the doses of 5, 15 and 50 mg/kg/day. The animals from the Control group were treated with Propylene glycol. Four groups of treatment were formed.
The main results are given below:
- No mortality was recorded
- No treatment-related clinical signs were recorded
- During the treatment period (days 6 to 19 of pregnancy) lower body-weight gain (%) and food consumption were recorded at the doses of 50 and 15 mg/kg/day. No effects on body growth or food intake were recorded among pregnant females treated at 5 mg/kg/day
- In the observations derived from hysterectomy, there were no relevant test-item changes.
- The skeletal examination of the fetuses showed a slightly increased incidence of medially thickened/kinked ribs, misaligned sternebral ossification sites and an increase in the ossified cervical vertebral centra at the dose of 50 mg/kg/day compared to the control group.
- The external and visceral examination of the fetuses showed the presence of major abnormalities in one fetus (no. 4 from the litter 50) at the dose of 5 mg/kg/day and two fetuses (no. 1 from litter 75 and no. 4 from litter 70) at the dose of 15 mg/kg/day. No external or visceral major findings were recorded at 50 mg/kg/day. Despite the fact that the external and visceral abnormalities recorded were observed in the groups treated with the test item, the incidence and distribution recorded could be considered not dose-related.

Based on the results of this study, the dose of 50 mg/kg/day is considered the dose with No Observed Adverse Effect Level (NOAEL) for pregnant females. The slight lower bodyweight increase recorded at this dose could be considered as an indication of a minimal toxic effect.

 

With respect to the effects on embryofetal development, the dose of 50 mg/kg/day is considered the dose with No Observed Adverse Effect Level (NOAEL). There was no apparent relationship in some differences recorded at the dose of 15 mg/kg/day.

Despite the presence of abnormalities at external and visceral examination at the low and intermediate doses, there was no evidence that the test item, administered by oral gavage up to 50 mg/kg/day, could significantly increase the occurrence of teratogenic effects. This dose could be considered the No Observed Adverse Effect Level (NOAEL).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

9 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

modern OECD 414 study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

To be compliant with the REACH Regulation, two pre-natal developmental toxicity studies need to be submitted for a substance registered under REACH Annex X.

For the registered substance (PACM) an OECD 414 with rats is available [Envigo, 2016], but an OECD 414 using a second species is not. However, a GLP guideline study (OECD 414, rabbit) of the structural analogue DMDC (CAS 6864-37-5) is on hand. [BASF, 2017]

PACM and DMDC are considered read-across analogues based on structural similarity and similar physico-chemical and toxicological properties. The common structural features of the two substances are: a common functional primary amine group; the amines are bound to a cyclic aliphatic organic substituent; there are no elements other than carbon, hydrogen and nitrogen; identical structures except for a methyl group on each cyclohexane, ortho to the amine on DMDC similar molecular weights, both under 500 daltons, qualifying as “low molecular weight” compounds. A read-across approach according to Regulation (EC) No 1907/2006 (REACH) Annex XI 1.5 and following ECHA Read-Across Assessment Framework, RAAF (2015) is attached in the respective IUCLID chapters.

PACM is registered under REACH Annex X with identified data gaps at the endpoint toxicity to reproduction. A study to check for fertility influencing effects as well as a study for pre-natal developmental toxicity (second species) are missing. However, these data are available for the analogue chemical DMDC. For this purpose, those data will be used as a source to fill existing data gaps (e.g. OECD 414 study, second species).

 

In a pre-natal developmental toxicity study (OECD 414) in pregnant female Wistar rats, PACM was administered once daily by oral gavage to three treatment groups of twenty animals each from day 6 to day 19 of gestation at dose levels of 5, 15 and 50 mg/kg bw. A control group of 22 females were administered with vehicle (propylene glycol) alone. The treatment with the test item resulted in no mortalities. All the dams survived till the scheduled sacrifice. No test item related clinical signs were observed in any of the pregnant female animals of low, intermediate and high dose groups as well as in control group. The body weight, body weight gain (%) and feed consumption during gestation period in all treatment groups were comparable with control group. No treatment-related effects were observed in reproduction parameters. The external and visceral variations observed in fetuses were not statistically significant. Therefore, these findings were considered as incidental findings and not test item related. Based on the study findings, it is considered that the test item is not a developmental toxicant in rats and the NOAEL of the test item can be fixed as 50 mg/kg bw, under the experimental conditions. The NOAEL for maternal toxicity was considered as 50 mg/kg bw. [Envigo, 2016]

In comparison, in a pre-natal developmental toxicity study on DMDC in rats, the animals were treated orally via gavage from day 6 to 19 post coitum inclusive with doses of 0, 5, 15 or 45 mg/kg bw. The test substance was prepared in 0.5% aqueous carboxymethylcellulose (OECD 414). Clear maternal toxicity was observed at the high dose level of 45 mg/kg bw, especially with regards to corrected body weight gain (-44%) and macroscopic findings (liver) of the dams. At the mid dose (15 mg/kg bw) maternal toxicity was less pronounced. There were no substance related effects with respect to gestational parameters. No external or soft tissue findings in fetuses were noted at all doses. A slight but significant retardation of ossification of the skull bones occurred only at the highest dose. The NOAEL for maternal toxicity was 5 mg/kg bw and 15 mg/kg bw with respect to fetotoxicity. The NOAEL for teratogenicity was 45 mg/kg bw, the highest dose tested. [BASF, 2019]

 

Both substances were tested in OECD 414 pre-natal developmental toxicity studies on rats using a very similar dose setting with the result that none of the available data give any hint on a potential of PACM as well as DMDC to influence the offspring’s development. These both studies were used a bridging studies.

It is assumed that the health effects may be predicted from the available data for the source substance at this specific endpoint: currently, there is no pre-natal developmental toxicity study (OECD 414) on the second species with PACM available. However, the read across substance DMDC was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28) at dose levels of 1, 3 and 9 mg/kg bw. Analyses confirmed the correctness of the prepared concentrations, their homogeneous distribution, and the stability of the test substance in the vehicle CMC (0.5%). Generally, clinical observations including food consumption and body weight gain revealed no toxicologically relevant effects at the low dose of 1 mg/kg bw. At the mid- and the high dose (3 and 9 mg/kg bw) signs of maternal toxicity were observed such as a dose dependent reduction of body weights/body weight gain, and reduced food consumption in the high-dose group, only. No differences of toxicological relevance between the control and the treated groups (1, 3 and 9 mg/kg bw) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and post-implantation loss. Similarly, no adverse effect of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose. Overall, there were no toxicologically relevant adverse effects of the test substance on fetal morphology at any dose. [BASF, 2017]

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available information are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available experimental information, the registered substance is not classified for toxicity to reproduction or developmental toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.